Acidic organelles are present in all cells and tissues of mammalian, plant, yeast and fungal cells, except red blood cells. Lysosomes are an example of an acidic cytoplasmic organelle that have been found to be involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. To date, more than 50 acidic hydrolytic enzymes have been identified that are involved in ordered lysosomal degradation of proteins, lipids, carbohydrates and nucleic acids. Functional deficiencies in these lysosomal enzymes, however, are indicative of a number of disease states.
As a group, these diseases are among the most prevalent genetic abnormalities of humans. Gaucher disease, Sandhoff disease, Krabbé disease, and Tay-Sachs syndrome comprise the majority of patients in this category and are categorized as sphingolipidoses in which excessive quantities of un-degraded fatty components of cell membranes accumulate due to mutations of specific catabolic enzymes that normally localize in the lysosomes to degrade such cellular components.
Without being bound by theory, it is thought that such genetic mutations may result in improper folding of these catabolic enzymes. Endoplasmic reticulum (ER) associated degradation serves as a “quality control” system for ensuring that only properly folded and assembled proteins are transported out of the ER for further maturation, while improperly folded proteins are retained for subsequent degradation (Hurtley S M, Helenius A (1989) Ann. Rev. Cell Biol. 5:277-307.). Accordingly, the disease state caused by such mutations may be a result of decreased enzyme stability, increased retention and degradation in the ER or impaired trafficking of the enzyme to the lysosome.
The therapeutic options for treating these diseases are relatively limited: in fact, there are currently no available therapies for many of these disorders. To date, therapeutic efforts have mainly focused on strategies for augmenting enzyme concentrations by providing large quantities of the enzyme (Enzyme Replacement Therapy, ERT) to compensate for the underlying defect (Grabowski, G A, Hopkin, R J (2003) Ann Rev. Human Genet. Genom. 4: 403). This type of therapy, however, has a number or drawbacks, including the inability of the administered protein to cross the blood-brain barrier where much of the neurological damage in these diseases can occur. Thus far, use of ERT has been largely unsuccessful in improving central nervous system manifestations for many of the lysosomal storage diseases, putatively due to difficulty in penetrating the blood-brain barrier.
Pharmacological chaperone therapy (PCT) has emerged as a possible new treatment option for diseases caused by improper protein folding or mis-trafficking. PCT relies on the ability of pharmacological chaperones (PCs) to bind to a mutant enzyme after it is made in the ER and promote a correctly folded conformation of the target mutant protein, thereby enabling it to meet the quality control standards in the ER and rescue it from degradation in the ER, and/or Golgi and restore trafficking to the trans-Golgi and lysosome.
Recent evidence has shown that accumulation of unprocessed compounds in cells results from low levels of functional enzymes, and not from low intrinsic catalytic enzyme activity of the low level of enzymes available. This indicates that the improperly folded enzymes retain sufficient functionality to remove or alleviate disease symptoms if they can simply avoid degradation by cellular quality control systems and thus supports the feasibility of PCT in treatment.
Although somewhat counterintuitive, enzyme competitive inhibitors can act as good pharmacological chaperones and increase the steady-state lysosomal levels of active enzymes through this rescuing mechanism. Once the inhibitor aids the enzyme in avoiding degradation in the ER, the inhibitor is eventually displaced from the active site, releasing the enzyme to conduct its intended catabolic activity in the lysosome.
Among the most common pharmacological chaperones developed to date are iminoalditols; imino-analogs of the sugar which the enzyme acts upon. Miglustat (OGT 918, N-butyldeoxynojirimycin) is one such competitive inhibitor that is used primarily to treat Type I Gaucher disease (GDI). Miglustat is an imino sugar, a synthetic analogue of D-glucose that contains a short-chain alkyl function on the imino nitrogen that promotes binding and bioavailability. It is one of the only small molecule pharmacological chaperones in clinical use. As a pharmacological chaperone, miglustat functions by helping promote correct folding of mutant enzymes and thereby bypass the degradation mechanisms located in the ER.
N-Alkyl iminoalditols, such as miglustat, or similar galactose, fucose, iduronate or mannose derivatives have also found use in combination with Enzyme Replacement Therapy (ERT) protocols. By administering the enzyme already coordinated with the inhibitor bound to the active site, intracellular levels of enzymes have been increased. To date, however, the activity of such iminoalditols for efficacy in treatment of the lysosomal storage disorders or allied diseases has been largely unsatisfactory. Without being bound by theory, such unsatisfactory results are likely due to the inability to concentrate sufficient amounts of miglustat in target organelles within the diseased cell.
Substrate reduction therapy (SRT) has also been developed as another alternative treatment option for these diseases. By inhibiting the initial biosynthesis of a precursor compound at an earlier metabolic step, it is postulated that the buildup of glycolipids or other biological compounds due to the defective enzyme will be abated. The therapeutic effect of substrate reduction therapy depends upon the presence of residual hydrolytic activity towards any accumulated substrates.
This approach has been used in the treatment of Gaucher disease through the inhibition of uridine diphosphate glucosylceramide transferase, the enzyme responsible for initial formation of the glucosyl compound that accumulates in Gaucher disease. One candidate for SRT is miglustat which is a known inhibitor of the enzyme glucosylceramide synthase that catalyzes the first step in the biosynthesis of glycosphingolipids (GSL), i.e., the formation of glucosylceramide (GlcCer). By reducing the formation of GlcCer, a decreased biosynthesis of more complex GSL is affected (Cox et al, (2000) “Novel oral treatment of Gaucher's disease with N-butyldeoxynojirimycin (OGT 918) to decrease substrate biosynthesis.” Lancet 355:1481). To date this approach has also resulted in unsatisfactory results which are likely due to the inability to concentrate sufficient amounts of miglustat in target organelles within the disease cell.
In addition to the lysosomal storage disorders, a large and diverse number of diseases are now recognized as conformational diseases that are caused by adoption of non-native protein conformations, leading to retardation of proteins in the ER and ultimate degradation (Kuznetsov et al, N. Engl. J. Med. 1998; 339:1688-1695: Thomas et al, Trends Biochem. Sci. 1995; 20:456-459; Bychkova et al., FEBS Lett. 1995; 359:6-8; Brooks, FEBS Lett. 1997; 409:115-120).
Small molecule pharmacological chaperones have been shown to rescue expression of mutant proteins other than enzymes. For example, small synthetic compounds were found to stabilize the DNA binding domain of mutant forms of the tumor suppressor protein p53, thereby allowing the protein to maintain an active conformation (Foster et al., Science 1999; 286:2507-10). Synthesis of receptors has been shown to be rescued by small molecule receptor antagonists and ligands (Morello et ai. J. Clin. Invest. 2000; 105:887-95; Petaja-Repo et al., EMBO J. 2002; 21:1628-37.). Even pharmacological rescue of membrane channel proteins and other plasma membrane transporters has been demonstrated using channel-blocking drugs or substrates (Rajamani et al., Circulation 2002; 105:2830-5; Zhou et al., J. Biol. Chem. 1999; 274:31123-26; Loo et al., J. Biol. Chem 1997; 272: 709-12). Also, pharmacological chaperones have also been identified that can act to rescue the mutant transmembrane regulator protein associated with cystic fibrosis, the ΔF508-CFTR protein, from ER degradation.
Pharmacological chaperones have also been shown to stabilize wild-type proteins, resulting in their enhanced production and stability. As one example, it has been demonstrated that 1-deoxygalactonojirimycin is able to increase the amount and activity of α-Gal A in COS-7 cells transfected with a vector coding the α-Gal A sequence. The Pharmacological chaperone is able to rescue the overexpressed enzyme, which is otherwise retarded in the ER quality control system, because overexpression and over production of the enzyme in the COS-7 cells exceeds the capacity of the system and leads to aggregation and degradation (U.S. patent application Ser. No. 10/377,179, filed Feb. 28, 2003).
In all cases the efficacy of pharmacological chaperone treatments is limited by the ability to deliver such molecules to the appropriate target organelle in sufficient quantities to produce a clinically useful result. Accordingly there exists a long-felt need in the art to create a system for the targeted delivery of such compounds.
Peptide motifs that can be used to target proteins or even small molecules to various locations within cells are known in the art. For example, the nuclear targeting sequence from the SV40 large T antigen PKKKRKV (SEQ ID NO: 1) has been used to localize exogenously delivered macromolecular conjugates (Branden L J, Christensson B, Smith C I. (2001) Gene Ther. 8(1):84-87.) to live cells as well as recombinant proteins expressed after plasmid or viral DNA transfection/transduction (Dingwall C, Laskey R A. (1991) Trends Biochem. Sci. 16(12):478-481.).
Peptide localization motifs have also been described for organelles other than the nucleus. For example, the four amino acid sequence KDEL (SEQ ID NO:2) at the amino terminus of a protein is a well established ER-retention sequence (Muaro S, Pelham H R. (1987) Cell. 48(5):899-907.), while the carboxy-terminal sequence of amino acids containing the amino acid sequence SKL has been identified for peroxisomal targeting (Gould S G, Keller G A, Subramani S. (1987) J Cell Biol 105(6 Pt 2); 2923-2931)
These and other targeting sequences have also been used for fluorescent labeling of specific organelles in live cells as an orthogonal method to cell staining by conjugation to small molecule organic dyes (Eward H. W. Pap, Tobias B. Dansen Ruben van Summeren & Karel W. A. Wirtz (2001) Experimental Cell Research 265: 288-293). These peptide sequences are known to be actively transported into living cells by the method of retrograde transport (Johannes L, Tenza D, Antony C, Goud B, (19971 J. Biol. Chem. 272: 19554-19561; Majoul I V. Bastiaens P I, Siling H D (1996) J. Cell Biol. 133(4):777-789.)
While such targeting peptides have been used as research tools for localizing compounds of interest in specific organelles their potential for targeted delivery of therapeutic agents within living cells or tissues remains unexplored. Such targeting has the potential to greatly improve the efficacy of known small molecule pharmacological chaperone compounds as well as other drugs known to act in specific organelles within living cells.